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Clinical Chemistry 45: 1162-1167, 1999;
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(Clinical Chemistry. 1999;45:1162-1167.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Detection of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis

Tian-Jian Chen1,2, Richard G. Boles2 and Lee-Jun C. Wong1,2,a

1 Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007.

2 Division of Medical Genetics, Children's Hospital Los Angeles, and Department of Pediatrics, University of Southern California, School of Medicine, Los Angeles, CA 90027.
a Address correspondence to this author at: Institute for Molecular and Human Genetics, 3800 Reservoir Rd. NW, Suite 4000, Georgetown University Medical Center, Washington, DC 20007. Fax 202-784-1770; e-mail wonglj{at}gunet.georgetown.edu

Background: A unique requirement for the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence variations before further testing (e.g., sequencing) is performed. We evaluated the potential utility of temporal temperature gradient gel electrophoresis (TTGE) for these purposes in patients with suspected mtDNA mutations.

Methods: DNA samples were selected from patients with known mtDNA mutations and patients suspected of mtDNA disorders without detectable mutations by routine analysis. Six regions of mtDNA were PCR amplified and analyzed by TTGE. Electrophoresis was carried out at 145 V with a constant temperature increment of 1.2 °C/h. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR/allele-specific oligonucleotide or PCR/restriction fragment length polymorphism analysis.

Results: In the experiments using patient samples containing various amounts of mutant mtDNA, TTGE detected as little as 4% mutant heteroplasmy and identified heteroplasmy in the presence of a homoplasmic polymorphism. In 109 specimens with 15 different known mutations, TTGE detected the presence of all mutations and distinguished heteroplasmic mutations from homoplasmic polymorphisms. When 11% of the mtDNA genome was analyzed by TTGE in 104 patients with clinically suspected mitochondrial disorders, 7 cases of heteroplasmy ({approx}7%) were detected.

Conclusions: TTGE distinguishes heteroplasmic mutation from homoplasmic polymorphisms and appears to be a sensitive tool for detection of sequence variations and heteroplasmy in patients suspected of having mtDNA disorders.© 1999 American Association for Clinical Chemistry




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